Analytical Performance Evaluation of Automated Coagulation Analyzer CP3000 for Routine and Special Coagulation Assays.

CP3000 coagulation analyzer is a high-throughput, fully automated coagulation analyzer. The objective of this study was to evaluate the analytical performance of CP3000 coagulation system for general and special coagulation analyses. Quality control materials and patient samples were used to evaluate the analytical performance of CP3000 coagulation system. Precision, carryover, linearity, comparability with ACL-TOP 700 coagulation system, and verification of reference range were evaluated or performed according to Clinical and Laboratory Standards Institute guidelines. Within-run and between-run precisions were below 5% for both normal and abnormal ranges. There was no detectable carryover. The linearity of antithrombin and fibrinogen were excellent. The comparability between CP3000 and ACL-TOP 700 coagulation systems was acceptable except for activated partial thromboplastin time and thrombin time due to differences in reagent composition. Reference ranges proposed by the manufacturer were verified to be acceptable. CP3000 coagulation system is a reliable system that can be used to perform routine and special coagulation tests rapidly and accurately. Because of its small footprint as an additional advantage, the implementation of CP3000 coagulation system can be efficient in hospital laboratories of various sizes.

Point-of-care testing for emergency assessment of coagulation in patients treated with direct oral anticoagulants.

BACKGROUND: Point-of-care testing (POCT) of coagulation has been proven to be of great value in accelerating emergency treatment. Specific POCT for direct oral anticoagulants (DOAC) is not available, but the effects of DOAC on established POCT have been described. We aimed to determine the diagnostic accuracy of Hemochron® Signature coagulation POCT to qualitatively rule out relevant concentrations of apixaban, rivaroxaban, and dabigatran in real-life patients. METHODS: We enrolled 68 patients receiving apixaban, rivaroxaban, or dabigatran and obtained blood samples at six pre-specified time points. Coagulation testing was performed using prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (aPTT), and activated clotting time (ACT+ and ACT-low range) POCT cards. For comparison, laboratory-based assays of diluted thrombin time (Hemoclot) and anti-Xa activity were conducted. DOAC concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Four hundred and three samples were collected. POCT results of PT/INR and ACT+ correlated with both rivaroxaban and dabigatran concentrations. Insufficient correlation was found for apixaban. Rivaroxaban concentrations at <30 and <100 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.0 and ≤1.1 and ACT+ POCT ≤120 and ≤130 s. Dabigatran concentrations at <30 and <50 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.1 and ≤1.2 and ACT+ POCT ≤100 s. CONCLUSIONS: Hemochron® Signature POCT can be a fast and reliable alternative for guiding emergency treatment during rivaroxaban and dabigatran therapy. It allows the rapid identification of a relevant fraction of patients that can be treated immediately without the need to await the results of much slower laboratory-based coagulation tests. TRIAL REGISTRATION: Unique identifier, NCT02371070 . Retrospectively registered on 18 February 2015.

Thrombin generation test in children and adolescents with chronic liver disease.

INTRODUCTION: The aim of this study was to evaluate if real-time thrombin generation assay provides additional information to assess the hemostatic balance in children with liver disease as compared to routine coagulation tests. PATIENTS AND METHODS: Sixty-three children with chronic liver disease were enrolled at our tertiary referral center for pediatric hepatology, eight whose routine coagulation tests gave abnormal results (Group A) and 55 whose test results were normal (Group B). Abnormal routine coagulation test was defined as at least one of the following: international normalized ratio≥1.4, activated partial thromboplastin time>44 sec., fibrinogen<1.5 g/L. Platelet-poor plasma was analyzed with the fluorogenic Calibrated Automated Thrombogram to test for thrombin generation, including endogenous thrombin potential. Further, routine coagulation tests and plasma levels of pro- and anticoagulant factors were measured. Twenty age-matched children without liver disease served as controls. RESULTS: The endogenous thrombin potential in the 55 patients with normal routine coagulation tests was not significantly different from that in controls. Group A had significantly lower levels not only of procoagulant factors (II, V, VII, X) but simultaneously also of the anticoagulant factors antithrombin, protein S free, and protein C. These patients had a reduced endogenous thrombin potential compared to Group B, in agreement with their routine coagulation test results. CONCLUSION: Thrombin generation analysis seems to give information on the hemostatic balance consistent with routine coagulation test results in children with liver disease. Further development and clinical evaluation of the method are warranted.

Coagulation phenotypes in septic shock as evaluated by calibrated automated thrombography.

Sepsis induces alterations of coagulation suggesting both hypercoagulable or hypocoagulable features. The result of their combination remains unknown, making it difficult to predict whether one prevails over the other. Thrombin generation tests (TGTs) stand as an interesting tool to establish an integrative phenotype of coagulation. It has been reported that septic patients display a hypocoagulable trait using TGT. However, protein C (PC) system response was not evaluated. We aimed at describing the thrombin generation profile in patients with septic shock under conditions that are sensitive to PC system to evaluate the net results of coagulation abnormalities and to determine whether hypercoagulable or hypocoagulable traits coexist within a given individual. Thrombin generation was studied in plasma from patients presenting with septic shock at diagnosis and 6 h after a conventional therapeutic management using calibrated automated thrombography with or without thrombomodulin (TM) addition. Patients exhibit clear alterations of TGT that present as both consumption-related hypocoagulability (evidenced without TM addition) but also hypercoagulability by decreased sensitivity to the PC system evidenced with TM addition. No difference could be demonstrated between survivors and nonsurvivors at Day 28, but patients who do not respond to therapeutics at 6 h seem to be more hypercoagulable. More importantly, if our results evidence heterogeneity between patients, we show that alterations of coagulation result in an equilibrium in the majority of patients, thus suggesting "normocoagulability"; but, in the presence of a biological imbalance between baseline thrombin generation and sensitivity to TM, the global effect mostly tends toward hypercoagulability. Thus, TGT may help identify distinct biological coagulation phenotypes in the complex alterations induced by sepsis.

The effect of the REG2 Anticoagulation System on thrombin generation kinetics: a pharmacodynamic and pharmacokinetic first-in-human study.

The REG2 Anticoagulation System consists of pegnivacogin, a subcutaneously administered aptamer factor IXa inhibitor, and its intravenous active control agent, anivamersen. Its effect on thrombin generation is unknown. A prospectively designed thrombin generation study was conducted within the phase 1 ascending dose study of REG2 to assess the effect of REG2 on thrombin generation kinetics. A total of 32 healthy volunteers were recruited into four cohorts of ascending dose pegnivacogin for the phase 1 study. In this pre-specified substudy, blood samples were drawn in the presence or absence of corn trypsin inhibitor at specified times within each dosing cohort. Thrombin generation was initiated with tissue factor and thrombin generation kinetics were measured using the Calibrated Automated Thrombogram (CAT). REG2 attenuated thrombin generation in a dose-dependent manner. All parameters of the CAT assay, except for lag time, showed a dose and concentration-dependent response to pegnivacogin [time to peak thrombin generation (PTm), endogenous thrombin potential, peak thrombin generation, and velocity index (VIx)]. Reversal of the effect of pegnivacogin with anivamersen demonstrated restoration of thrombin generation without rebound effect. This first-in-human study of the effect of the REG2 Anticoagulation System on thrombin generation demonstrates concentration-dependent suppression of thrombin generation that is reversible without rebound effect, as measured by the CAT assay.

Thrombin generation using the calibrated automated thrombinoscope to assess reversibility of dabigatran and rivaroxaban.

The new direct-acting anticoagulants such as dabigatran and rivaroxaban are usually not monitored but may be associated with haemorrhage, particularly where renal impairment occurs. They have no effective "antidotes". We studied 17 patients receiving dabigatran 150 mg twice daily for non-valvular atrial fibrillation and 15 patients receiving rivaroxaban 10 mg daily for the prevention of deep venous thrombosis after hip or knee replacement surgery. We assessed the effect of these drugs on commonly used laboratory tests and Calibrated Automated Thrombogram (CAT) using plasma samples. We also assessed effects in fresh whole blood citrated patient samples using thromboelastography on the TEG and the ROTEM. The efficacy of nonspecific haemostatic agents prothrombin complex concentrate (PCC), Factor VIII Inhibitor By-passing Activity (FEIBA) and recombinant activated factor VII (rVIIa) were tested by reversal of abnormal thrombin generation using the CAT. Concentrations added ex vivo were chosen to reflect doses normally given in vivo. Dabigatran significantly increased the dynamic parameters of the TEG and ROTEM and the lag time of the CAT. It significantly reduced the endogenous thrombin potential (ETP) and reduced the peak height of the CAT. Rivaroxaban did not affect the TEG and ROTEM parameters but did increase the lag time and reduce ETP and peak height of the CAT. For both drugs, these parameters were significantly and meaningfully corrected by PCC and FEIBA and to a lesser but still significant extent by rFVIIa. These results may be useful in devising a reversal strategy in patients but clinical experience will be needed to verify them.

Monitoring complex formation in the blood-coagulation cascade using aptamer-coated SAW sensors.

Specific binding of the anticoagulants heparin and antithrombin III to the blood clotting cascade factor human thrombin was recorded as a function of time with a Love-wave biosensor array consisting of five sensor elements. Two of the sensor elements were used as references. Three sensor elements were coated with RNA or DNA aptamers for specific binding of human thrombin. The affinity between the aptamers and thrombin, measured using the biosensor, was within the same range as the value of K(D) measured by filter binding experiments. Consecutive binding of the thrombin inhibitors heparin, antithrombin III or the heparin-antithrombin III complex to the immobilized thrombin molecules, and binding of a ternary complex of heparin, anithrombin III, and thrombin to aptamers was evaluated. The experiments showed attenuation of binding to thrombin due to heparin-antithrombin III complex formation. Binding of heparin activated the formation of the inhibitory complex of antithrombin III with thrombin about 2.7-fold. Binding of the DNA aptamer to exosite II appeared to inhibit heparin binding to exosite I.